G protein-coupled receptors (GPCRs) constitute the largest gene family in the human genome and accumulate the most successful biochemical targets for therapeutic drugs. The natural ligands of about 140 GPCRs are unknown and they are thus called "orphan" receptors. Identification of these natural ligands forms the crucial step to start studying their function and holds the potential for development of novel pharmaceutical drugs. We have developed a strategy to isolate such natural ligands by expressing the cloned receptors in suitable cell lines and monitoring activation of the receptors after adding fractions of tissue extracts. This novel strategy will ultimately allow us to purify the active molecules and determine their structure. In this application, we propose to apply this highly innovative technique to isolate, identify and characterize the natural ligand of the orphan receptor bombesin subtype-3 (BRS-3) that is still elusive. BRS-3 receptor is expected to play an important role in integrating and modulating the complex physiology underlying feeding as evidenced by the fact that mice devoid of BRS-3 receptor are hyperphagic, hypometabolic and develop an obese phenotype. This receptor does not bind the two known mammalian bombesin counterparts of the frog peptide neuromedin B and gastrin-releasing peptide that bind to the other two bombesin receptor subtypes (BB-1 and 2). Following the isolation of the natural ligand, we will characterize the pharmacological effects, study the Subsequently, we propose to study the BRS-3 ligand in a behavioral feeding assay in order to determine its mode of action. The BRS-3 ligands will prove as an invaluable tool to answer the functional significance and understand the physiological role of the BRS-3 system in the regulation of energy balance.